Intracellular trafficking and processing of amyloid precursor protein (APP) to generate amyloid beta protein (Abeta) was studied.1.Antibodies against specific protein in intracellular compartments : We were successful in raising specific antibodies against intracellular compartment specific protein, ERGIC53, galactosyltransferase, cathepsin D.2.APP trafficking in cultured cells : APP and Abeta immunoreactivities were observed in cell surface, and vesicular structures in cytoplasm. Western blot analyzes showed 50,20 kDa amyloidogenic fragments, and 4 kDa, apparently Abeta, in microsomal fraction.3.Effect of brefeldin A and monensin on APP processing : Brefeldin A and monensin are able to inhibit protein trafficking from endoplasmic reticulum (ER) to Golgi apparatus, and from Golgi apparatus to another cellular compartments. Under brefeldin A treatment, numerous vacuoles appeared in the cytoplasm of the cultured cells, and the vacuoles were immunolabeled with anti-ERGIC53. With monensin
… Moretreatment, vacuoles appeared in perinuclear cytoplasm, and were stained with anti-APP and anti-Golgi antibodies. Western blot analysis showed reduced intensity of Abeta 4 kDa band.4.APP trafficking in Hela cells transfected with wild type APP770, and Swedish mutant cDNA : Wild type APP770 and Swedish mutant APP were overexpressed in cultured HeLa cells. APP immunoreactivities appeared to increase in ER,cell surface, and vesicles in the cytoplasm of these transfected cells. APP mRNA and Swedish type mRNA were determined with Northern ELISA,and turned out to increase about 3 to 6 fold compared to plasmid controls.Our data provide new evidence indicating that APP may process to generate Abeta either in ER,or lysosomes, or both compartments may be involved.3.Monensin並びにBrefeldinAのAPPの細胞内輸送への影響:BrefeldinA、Monensinは、ERおよびGolgiからの蛋白輸送を阻害する。BrefeldinAを添加すると、核周囲に空胞が見られERGIC53抗体で染色され、4kDa前後のAβが検出された。Monensinの場合には、Golgi周囲に空胞が出現し、APP抗体並びにGolgi抗体で染色され、4kDaのAβバンドが減少した。4.APPcDNAの強制発現と細胞内輸送経路:野生型(APP770)及び、変異型(Swedish型)cDNAをHeLa細胞に強制発現させた。1)蛍光顕微鏡による解析:遺伝子導入後、APPの免疫反応はER、ならびに細胞表面、小胞様構造で増強した。2)細胞内局在に関する分子生物学的生化学的検討:遺伝子導入後mRNAは3-6倍発現した。4kDaのAβバンドは若干増強していた。以上の所見はGolgi前後の細胞内小器官、つまりER、あるいはリソソームの一方か両方において、APPからAβが生成されることを示唆する重要な所見と考えられる。 Less